Ataxin-2 regulates RGS8 translation in a new BAC-SCA2 transgenic mouse model

Abstract

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder with progressive degeneration of cerebellar Purkinje cells (PCs) and other neurons caused by expansion of a glutamine (Q) tract in the ATXN2 protein. We generated BAC transgenic lines in which the full-length human ATXN2 gene was transcribed using its endogenous regulatory machinery. Mice with the ATXN2 BAC transgene with an expanded CAG repeat (BAC-Q72) developed a progressive cellular and motor phenotype, whereas BAC mice expressing wild-type human ATXN2 (BAC-Q22) were indistinguishable from control mice. Expression analysis of laser-capture microdissected (LCM) fractions and regional expression confirmed that the BAC transgene was expressed in PCs and in other neuronal groups such as granule cells (GCs) and neurons in deep cerebellar nuclei as well as in spinal cord. Transcriptome analysis by deep RNA-sequencing revealed that BAC-Q72 mice had progressive changes in steady-state levels of specific mRNAs including Rgs8, one of the earliest down-regulated transcripts in the Pcp2-ATXN2[Q127] mouse line. Consistent with LCM analysis, transcriptome changes analyzed by deep RNA-sequencing were not restricted to PCs, but were also seen in transcripts enriched in GCs such as Neurod1. BAC-Q72, but not BAC-Q22 mice had reduced Rgs8 mRNA levels and even more severely reduced steady-state protein levels. Using RNA immunoprecipitation we showed that ATXN2 interacted selectively with RGS8 mRNA. This interaction was impaired when ATXN2 harbored an expanded polyglutamine. Mutant ATXN2 also reduced RGS8 expression in an in vitro coupled translation assay when compared with equal expression of wild-type ATXN2-Q22. Reduced abundance of Rgs8 in Pcp2-ATXN2[Q127] and BAC-Q72 mice supports our observations of a hyper-excitable mGluR1-ITPR1 signaling axis in SCA2, as RGS proteins are linked to attenuating mGluR1 signaling.

Fig 1. Generation of a BAC-SCA2 transgenic mouse model.

(A) Schematic representation of the modified 169 kb BAC containing the entire 150 kb human ATXN2 genomic locus, plus 16 kb 5’-flanking and 3 kb 3-’ flanking genomic regions. For the BAC-Q72 line, the BAC was engineered to replace the endogenous ATXN2 exon-1 CAG22 with CAG72 repeats. (B) RT-PCR analyses revealed expression of BAC-Q22 or BAC-Q72 in mouse CNS and non-CNS tissues. Synthesized cDNAs from mouse tissues were subjected to RT-PCR analysis using human ATXN2 specific primers and CAG primers as indicated. The Gapdh gene was amplified as an internal control. (C) The BAC-Q22 transgene is expressed at higher levels than the BAC-Q72 transgene. Quantitative RT-PCR analyses of cerebellar RNA from wild-type and transgenic mice measuring endogenous murine and human ATXN2 transgene. Note that direct comparison with expression levels of murine ATXN2 is not possible owing to different primer sets. Three animals per group were used for these analyses. (D) Western blot analyses of human ATXN2 protein in BAC-Q22 or BAC-Q72 mouse cerebella. Protein extracts from wild-type and transgenic mouse cerebella were subjected to Western blot analyses using ATXN2 or 1C2 mAbs. Two animals per group were used for Western blot analyses. Representative Western blots of three independent experiments are shown. β-actin was used as loading control.

Fig 2. BAC derived- hATXN2 mRNA is identified in multiple layers of the cerebellum and deep cerebellar nuclei.

Expression of transgenic hATXN2 and murine Atxn2 mRNAs in cerebellar fractions isolated by Laser Capture Microdissection (LCM): ML, molecular layer; PC, Purkinje cell layer; GCL, granule cell layer; DN, dentate nucleus. (A-B) Quantitative RT-PCR analyses of transgenic hATXN2 (A) and endogenous mAtxn2 (B). (C-F) Relative enrichment of cell-type specific marker genes; Pcp2, Calb1, Neurod1 and Spp1 for each fraction as determined by qRT-PCR. The error bars indicate ± SD.

Fig 3. Motor phenotype of ATXN2 BAC transgenic mice on the accelerating rotarod.

(A) BAC-Q22 mice performed as well as wild-type mice at all ages. (B) BAC-Q72 mice performed significantly worse than wild-type littermates on the rotarod starting at 16 weeks of age. Data represent mean ± SEM of three trials on the test day (day 3). Number of animals tested are shown within the bars. Significance was determined using repeated measures ANOVA with post-hoc test correction. *p<0.05 and ***p<0.001.

Fig 4. PC morphology in BAC-Q22 and BAC-Q72 mice.

(A) Representative micrographs of calbindin-28k immunostaining of PCs in the cerebellum of BAC-Q22, BAC-Q72, and wild-type mice at 24 weeks of age. Note that reduction of calbindin immunoreactivity and disorganization of the PC layer are only observed in BAC-Q72 cerebella (see also S4B Fig). (B) Western blot analyses show reduction of Calb1 and Pcp2 protein in BAC-Q72 mouse cerebella compared with wild-type at 24 weeks of age. Two animals per group were used for these analyses and the blots represent three independent experiments.

Fig 5. Early expression changes of key cerebellar genes including several PC-specific genes measured by quantitative RT-PCR.

(A) No significant changes in BAC-Q22 mice compared with wild-type at 16 and 45 weeks of age. (B) In BAC-Q72 mice, a small reduction of Pcp2 mRNA levels is seen at 5 weeks, but significant reductions in three genes are only seen at 9 weeks. Reductions in expression of Grm1 occur late (weeks 24 and 36). Of note, mRNA levels of mouse Atxn2 remain unchanged throughout. Genes tested: human transgene (hATXN2), mouse Ataxin-2 (mAtxn2), calbindin 28-kDa (Calb1), PC protein 2 (Pcp2), glutamate receptor ionotropic delta-2 (Grid2) and metabotropic glutamate receptor 1 (Grm1). n: animal numbers for each genotype and age group are listed in brackets. Gene expression was normalized to beta-actin. Student’s two-tailed t-test compared expression in BAC transgenic mice with wild-type mice in each age group. *p<0.05, **p<0.01, ***p<0.001. Error bars represent ± SD.

Fig 6. Comparison of transcriptome changes in BAC-Q72 and Pcp2-ATXN2[Q127] mice.

(A) The Venn diagram of transcriptome changes using an FDR ≥15 and Log2 ratio of change ≥|0.3|. Class I transcripts are changed only in BAC-Q72 and class III transcripts changed only in Pcp2-ATXN2[Q127] cerebella. A total of 236 transcripts (class II) are significantly altered in both models. (B) Validation of six overlapping genes (class II) by qRT-PCR. Cerebellar RNAs from BAC-Q72 and WT littermates (both at 8 weeks of age) and Pcp2-ATXN2[Q127] and WT littermates (both at 6 weeks of age) show significant reductions of transcript expression. Genes tested are; Rgs8, Calb1, Pcp2, Purkinje cell protein 4 (Pcp4), Homer homolog 3 (Drosophila) (Homer3) and Centrosomal protein 76 (Cep76). Gene expression levels were normalized to beta-actin. Six animals from each group were used in this experiment. Data are means ± SD, *p<0.05 **p<0.01, ***p<0.001, Student t-test. (C) Fold change relation between RNA-seq data and observed experimental qRT-PCR data are tabulated.

Fig 7. Decreased steady-state levels of Rgs8 message and protein in BAC-Q72 mice.

(A) qRT-PCR analyses of cerebellar RNAs from wild-type and BAC-Q22 mice show unchanged Rgs8 levels, whereas BAC-Q72 mice show significant and progressive reduction of Rgs8 mRNA levels starting at 5 weeks of age. n: number of animals in each group. The data are means ± SD, **p<0.01, ***p<0.001. (B) Western blot analyses indicate reduction of Rgs8 steady-state levels in cerebella of BAC-Q72 mice, but no change in BAC-Q22 mice when compared with wild-type mice. The blot is a representative Western blot of 3 independently performed experiments with 2 animals each per BAC line. (C) SCA2 patient-derived LB cells demonstrate decreased RGS8 transcripts. Total RNAs were isolated from LB cell lines derived from two normal control individuals and two SCA2 patients and subjected to RT-PCR analysis using primers specifically amplifying the human ATXN2 CAG repeat. RT-PCR analyses indicate the expression of ATXN2 with expanded CAG repeats (46 or 52) (left panel). qRT-PCR analyses of synthesized cDNAs from LB cells show significant reduction of RGS8 in both SCA2-LB cell lines. The data represent mean ± SD, **p<0.01 (right panel).

Fig 8. Overexpression of mutant ATXN2 in human SH-SY5Y cells recapitulates down-regulation of in vivo steady-state levels of Rgs8 in BAC-Q72 mice.

Cells were transfected with plasmids encoding Flag-tagged cDNAs of human ATXN2 containing Q22 or Q58 or Q108 repeats. Forty-eight hrs post-transfection, cells were selected with hygromycin (40 µg/ml) for 5–7 days and hygromycin resistant cells were harvested as two aliquots. (A) Protein extracts were prepared from one aliquot and subjected to Western blot analyses to measure steady-state levels of RGS8. The blots were re-probed for β-Actin as an internal loading control. (B) Quantitative RT-PCR analyses of synthesized cDNAs from the other aliquot demonstrate moderate reduction of RGS8 mRNA in cells expressing Flag-ATXN2-Q108. The data are means ± SD, *p<0.05. (C) Mutant ATXN2 specifically induces decrease of RGS8 expression. MYC-tagged RGS8 cDNA including 5’ and 3’ UTRs was cloned under the transcriptional control of the CMV promoter and transfected into short-term hygromycin selected SH-SY5Y cell lines expressing Flag-tagged ATXN2-Q22, -Q58 or -Q108. Forty-eight hrs post-transfection, levels of exogenous RGS8 are significantly decreased in cells expressing ATXN2-Q58 or -Q108 compared with cells expressing wild-type ATXN2-Q22. To control for equal transfection, we monitored levels of GFP, which was expressed as an independent cassette in the plasmid. Blots were re-probed for β-actin as an internal loading control. The blot represents one of three independent experiments.

Fig 9. ATXN2 immunoprecipitates RGS8 mRNA and regulates RGS8 steady state levels in vitro.

(A) SH-SY5Y whole cell extracts expressing Flag-ATXN2-Q22 or Flag-ATXN2-Q108 were subjected to immunoprecipitation with a mAb to the Flag tag. After washing the beads with buffer (200 mM NaCl), bound protein-RNA complexes were eluted by Flag peptide competition. IP products were divided equally into two parts and subjected to Western blot and RT-PCR analyses to identify ATXN2 interacting proteins and RNAs. Western blot analyses of the eluted proteins show co-IP of PABPC1 and DDX6, known ATXN2 interactors. RT-PCR analyses of the second aliquot show that both Flag-ATAXN2-Q22 and Flag-ATAXN2-Q108 immunoprecipitate RGS8 mRNA but not GAPDH mRNA. ATXN2-Q108 shows differential binding toward RGS8 mRNA when compared with ATXN2-Q22. (B) Interaction of ATXN2 with RGS8 mRNA determined by qRT-PCR. Synthesized cDNAs from the second aliquot of IP products (A) were subjected to qRT-PCR analyses. Interaction of RGS8 mRNA with ATXN2-Q108 was significantly reduced when compared with ATXN2-Q22. Data are mean ± SD, n = 3 independent experiments. **p<0.01. (C-D) Mutant ATXN2 represses RGS8 synthesis in vitro. First, cDNA plasmids of LacZ (control) and Flag-tagged ATXN2-Q22 or -Q108 were added to rabbit reticulocyte lysate mixture and proteins synthesized for 2 hrs. RGS8 cDNA plasmid was added to each translational reaction with fresh rabbit reticulocyte lysate and incubated further for 4 hrs. The synthesized RGS8 protein from each translational product was analyzed by SDS-PAGE followed by Western blot analyses. ATXN2-Q108 reduces RGS8 synthesis significantly when compared with ATXN2-Q22 (C). Quantification of RGS8 on Western blots, data are mean ± SD, n = 3 independent experiments. **p<0.01, Student’s t-test) (D). The blot represents one of three independent immunoprecipitation experiments.